Isolation and identification of Rhizomucor pusillus from rhinofacial mucormycosis in a diabetic patient

Case presentation: Here we report a case of rhinofacial mucormycosis due to Rhizomucor pusillus in a 55-year-old diabetic female. She presented with diabetic ketoacidosis and nasal obstruction, nasal discharge and right-sided cheek swelling following sinus surgery, which had been performed at a private hospital 1 month previously. Endoscopic biopsy was performed and the sample was sent for histopathological examination and KOH wet mount, which showed broad, pauci-septate hyphae with right-angle branching. The tissue was inoculated onto Sabouraud dextrose agar and white mycelial growth was obtained which turned grey with age. Morphological identification confirmed it as Rhizomucor pusillus. In vitro antifungal susceptibility testing was performed by means of the microbroth dilution method according to CLSI Approved Standard M38-A. The isolate was found to be susceptible to amphotericin B, itraconazole and posaconazole but resistant to voriconazole and echinocandins. Functional endoscopic sinus surgery was performed and local necrotic tissue was debrided. The patient was put on liposomal amphotericin B, with a successful outcome.


Introduction
Mucormycosis is an invasive fungal infection caused by members of the order Mucorales.Rhino-orbito-cerebral mucormycosis accounts for one-third to one-half of all cases of mucormycosis (Roden et al., 2005).Various risk factors contribute to the development of mucormycosis, the most common being diabetes mellitus with ketoacidosis (Do ¨kmetas ¸et al., 2002).Genus Rhizopus is most commonly isolated from rhino-orbito-cerebral mucormycosis cases.Rhizomucor pusillus belongs to the genus Rhizomucor and family Mucoraceae.It is a thermophilic saprobic fungus mainly causing infection in immunocompromised patients (Ribes et al., 2002).Worldwide there were only 22 cases of R. pusillus infection reported before 2013.Only one case of R. pusillus infection in India was reported during 1990-2007 (Gomes et al., 2011).Rhinoorbito-cerebral mucormycosis caused by R. pusillus was found only in 9 % of cases.Immunocompromising conditions were present in 91 % of mucormycosis cases due to R. pusillus, haematological disorders and malignancies being the most common (73 %).Diabetes mellitus was present only in one instance.We here report the case of a 55-year-old diabetic female suffering from rhinoorbito-cerebral mucormycosis caused by R. pusillus.

Case report
A 55-year-old female patient presented with a 1 month history of nasal blockage, nasal discharge, watering of eyes, right-sided facial swelling and loosening of teeth.On examination, she had purulent discharge in the right nasal cavity with small polyps.Her right side upper teeth were mobile.The swelling was accompanied with pain and intermittent fever.But there was no history of nasal bleeding, facial weakness and numbness, vision loss or periorbital pain.There was also history of right-sided molar tooth pain 1 month previously.She underwent a Caldwell Luc operation at Omni hospital, Chandigarh.But 1 month later she developed swelling of the right cheek.She had recently been diagnosed with type II diabetes mellitus but was not having any treatment for diabetes mellitus.Her father and brother also had type II diabetes mellitus.She also had hypertension.There was no history of any corticosteroid intake, iron therapy, haematological malignancy, trauma, transplantation, dialysis or intravenous drug intake.Her blood sugar level (fasting) was 310 mg dl 21 , haemoglobin 12.6 g dl 21 , urea 12 mg dl 21 , creatinine 0.93 mg dl 21 and bicarbonate level 7 mmol l 21 .Her HIV status was negative.An NCCT scan of the paranasal sinus showed complete fracture of the right lateral part of both anterior and posterior right maxilla at the level of the alveolar recess.Opacification and hypertrophy of inferior turbinates was seen on both sides.There was also opacification of the left maxillary antrum and ethmoidal sinus.Endoscopic biopsy was performed and the material was sent to the Departments of Microbiology and Pathology of our hospital (Government Medical College Hospital, Chandigarh).

Mycological and histopathological investigations
A KOH wet mount of the biopsy specimen showed sparsely septate, broad, ribbon-like hyphae with right-angle branching suggestive of mucormycosis (Fig. 1a).A portion of biopsy material was cut into small pieces without homogenization and inoculated onto Sabouraud dextrose agar either supplemented with chloramphenicol and gentamicin or without antibiotics.One set of tubes was incubated at 22 u C and another at 37 u C.After 24 h of incubation, initially white floccose growth with low aerial tufts was obtained, which on further incubation turned grey within 2-3 days.The growth of R. pusillus has low aerial tufts compared with most other mucoraceous moulds.On lactophenol cotton blue examination, sporangiophores were branched irregularly and extensively.Internodal poorly formed rhizoids were present.
Histopathological examination of the biopsy material from the right maxillary sinus after haematoxylin and eosin (Fig. 2a), periodic acid-Schiff (Fig. 2b) and Gomori methenamine silver staining showed large areas of necrosis, inflammatory exudate and many pauci-septate broad fungal hyphae with right-angle branching conforming to the morphology of mucormycetes.

In vitro antifungal susceptibility testing
In vitro antifungal sensitivity was determined by the microbroth dilution method according to CLSI (2005).Inoculum suspensions were prepared from 7 day potato dextrose agar (PDA; Difco) cultures by adding sterile saline solution and lightly scraping the surface of mature colonies with a sterile cotton swab.The homogeneous conidial suspensions were then transferred to sterile tubes and the supernatants were adjusted spectrophotometrically to OD 530 0.15-0.17

Treatment
The patient's blood sugar was controlled with human insulin twice a day.Her hypertension was controlled with 25 mg atenolol twice a day.Functional endoscopic sinus

Outcome and follow-up
This treatment led to significant reduction in facial swelling and the patient's condition improved.She was discharged in good condition with advice for regular follow-up.

Discussion
In 1978, the genus Rhizomucor was established for Mucorlike fungi forming stolons and rudimentary rhizoids and expressing thermophilism (Ribes et al., 2002).Molecular phylogenetic studies based on nuclear small and large subunit rRNA gene sequences support the distinction of R. pusillus from Mucor species.Rhizomucor is commonly found contaminating air, soil and organic matter.Various species of Rhizomucor are known.R. pusillus, Rhizomucor miehei and Rhizomucor variabilis with two subspecies (R. variabilis var.variabilis and R. variabilis var.regularior) can cause mucormycosis in humans.R. variabilis was recently shown to be phylogenetically nested far from R. pusillus but within the clade Mucor (Gomes et al., 2011).R. variabilis var.variabilis has now been renamed as Mucor irregularis.R. pusillus is the most common species seen and has been detected in a variety of food items, including grains, seeds, nuts and beans.It is a thermophilic saprobic mucormycete with a wide distribution but it is not commonly associated with human disease (Hernanz et al., 1983).Its mode of transmission is by inhalation of spores and percutaneous introduction of spores into a susceptible host (Lu et al., 2009).Various studies in the literature have shown this species to be mostly associated with patients who are severely immunocompromised, especially those undergoing therapy for leukaemia and those who have uncontrolled diabetes mellitus (Germain et al., 1993).The pathogenicity of R. pusillus is due to its angioinvasive nature and thermotolerance, which presumably allow it to grow in febrile patients (De pauw, 2008).Various presentations include primary cutaneous disease with or without dissemination, pulmonary disease and sinusitis with or without orbital participation or involvement of the brain.The majority of cases of R. pusillus infection present as pulmonary or disseminated disease.Lungs were the most commonly affected organs; pulmonary lesions were seen in 16 cases (Kimura et al., 2009).In five of these cases infection was restricted to the lung, whereas in another ten cases lung involvement was a part of disseminated, cardiopulmonary, sino-pulmonary infection.In one of these cases, initial rhino-orbito-sinus infection spread to the brain and lung.Sinus-only involvement was seen in two cases and cutaneous infections were only found in two cases.Patients with isolated pulmonary infection had a better prognosis than patients with disseminated infection (survival rates 80 and 22 %, respectively).Nine cases of disseminated infection due to R. pusillus have been reported in the literature (Germain et al., 1993).The underlying diseases in these cases were acute leukaemia (n57) and non-Hodgkin's lymphoma with renal transplantation and aplastic anaemia (n51).The rhino-facial and rhino-orbito-facial R. pusillus infections were reported for an 11-year-old boy with acute leukaemia and a 38-year-old woman with diabetic ketoacidosis, respectively (Iwen et al., 2005).R. pusillus infection may also initially appear as a sino-orbital infection that rapidly invades the brain in patients with haematological malignancies.In our case report, the patient was diabetic and suffered from rhino-facial mucormycosis due to R. pusillus.Early diagnosis of R. pusillus infection often is feasible only in patients with accessible lesions who can tolerate a biopsy, which is required for histopathological identification and culture.R. pusillus has a temperature growth range of 20 to 60 u C. Morphological and biochemical properties help to differentiate R. pusillus from other mucormycetes.Mating studies are considered best for morphological identification of mucormycete species but they require maintenance of a library of testing strains.Zygospore production is useful for differentiating R. pusillus from R. miehei, as the former produces heterothallic zygospores.The higher tolerance of R. miehei to lovastatin can be useful for differentiating it from other species.DNA sequencing for comparison with known type strains in international databases is currently considered the best way to identify and classify mucormycete species.PCR and RFLP or other molecular methods have been used for R. pusillus identification in culture tissue, and these techniques may be more rapid and reliable than standard mycological identification (De Hoog et al., 2000).
The overall mortality rate for Rhizomucor infections is significantly lower than that seen with other mucoraceous moulds.Among 18 out of 22 patients who received antifungal treatment, 11 survived (Gomes et al., 2011).Surgical debridement in addition to liposomal amphotericin B at a minimum dose of 5 mg kg 21 day 21 remains the treatment of choice for mucormycosis (Cornely et al., 2014).Our patient was managed by surgical treatment and liposomal amphotericin B treatment.Timely diagnosis and treatment aided in her survival.